Optical spectroscopy for the detection of ischemic tissue injury

ABSTRACT

An optical method and apparatus is utilized to quantify ischemic tissue and/or organ injury. Such a method and apparatus is non-invasive, non-traumatic, portable, and can make measurements in a matter of seconds. Moreover, such a method and apparatus can be realized through optical fiber probes, making it possible to take measurements of target organs deep within a patient&#39;s body. Such a technology provides a means of detecting and quantifying tissue injury in its early stages, before it is clinically apparent and before irreversible damage has occurred.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/535,585, filed Jan. 8, 2004, and U.S. Provisional Application No.60/559,887, filed Apr. 5, 2004, both entitled, “Optical Spectroscopy forthe Detection of Ischemic Tissue Injury,” which is incorporated hereinby this reference.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the United States Department ofEnergy and the University of California for the operation of LawrenceLivermore National Laboratory.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a medical diagnostic for investigatingtissue components. More particularly, the present invention relates toan apparatus and a method that utilizes autofluorescence emission and/orpolarized elastic light scattering as an interrogation means forinvestigating tissue states for medical applications.

2. Description of Related Art

Transplant surgeons often face the difficult question of whether organsfrom a cadaveric donor are suitable for transplantation. It is currentlydifficult to quantify how much warm ischemic organ damage has occurred,especially if a donor has experienced significant hemodynamicinstability. Organ procurement in brain-dead and non-heart-beatingdonors can cause a variable amount of additional warm ischemia.Transplanting organs that have sustained significant pre-implantationwarm ischemic damage may leave the recipient with unacceptably poorallograft function and adversely affect graft and patient survival. Onthe other hand, as transplant waiting lists continue to grow, it becomesincreasingly important to maximize utilization of viable organs frommarginal donors.

Post transplant monitoring is currently the only reliable way ofassessing ischemic organ damage; unfortunately, at that stage the organis already in the recipient and may have to be removed if found to benonviable. Three fundamental questions regarding ischemia in atransplanted organ are apparent. First, how much ischemic damage has anorgan sustained? Second, how does the organ respond to interventionaimed at halting or reversing ischemic damage? Third, how will the organfunction after it is transplanted? Beneficial methods and apparatus foranalyzing organ ischemia and answering such questions is needed at anystage of transplantation, while the organ is still in the donor, afterit has been removed (during hypothermic preservation), while it is beingimplanted, and after reperfusion. Such methods and apparatus requirereal-time, substantially instantaneous results so that clinicaldecisions can be made in a timely fashion.

Background information on an existing approaches that addresses organdamage is described by Inman S., Osgood R., Plott W., et al., in“Identification of kidneys subjected to pre-retrieval warm ischemicinjury during hypothermal perfusion preservation,” Transplant Proc.,1998, pp. 42-46, and in “The non heart-beating donor,” by Kootstra G.,Kievet J K, Heineman E., Br Med Bull., (1997); 53 (4): 844, and in“Release of alpha-glutathione S-transferase (alpha GST) andpi-glutathione S-transferase (pi GST) from ischemic damaged kidneys intothe machine perfusate-relevance to viability assessment,” TransplantProc. (1997); 29 (8): 3591. However, such approaches are hindered byclinical practicality as well as by hypothermic preservation.

Background information on monitoring tissue viability is described andclaimed in U.S. Patent No. 2004/0054270 A1, entitled “Apparatus AndMethod For Monitoring Tissue Vitality Parameters,” issued Mar. 18, 2004to Pewzner et al., including the following, “Apparatus for monitoring aplurality of tissue viability parameters of a tissue layer element, inwhich two different illumination sources are used via a commonillumination element in contact with the tissue. One illumination sourceis used for monitoring blood flow rate and optionally flavoproteinconcentration, and collection fibers are provided to receive theappropriate radiation from the tissue. The other illuminating radiationis used for monitoring any one of and preferably all of NADH, bloodvolume and blood oxygenation state of the tissue element, and collectionfibers are provided to receive the appropriate radiation from thetissue.” However, such techniques from the above cited patent does notaddress detecting and quantifying ischemic tissue damage in itsincipient stages in accordance with the principles of the presentinvention.

Accordingly, a need exists for methods and apparatus that can detect andquantify tissue parameters in its early stages before injury isclinically apparent and before irreversible damage has occurred. Thepresent invention is directed to such a need.

SUMMARY OF THE INVENTION

Accordingly, the present invention is directed to a method for assessingtissue parameters, including: illuminating a tissue component regionwith a first substantially narrow spectral band source; illuminating thetissue component region with a second substantially narrow spectral bandsource, wherein the illuminated region by the first and said secondsubstantially narrow spectral band sources comprises an area of greaterthan about 1.5 mm²; detecting a respective autofluorescence emissionfrom the illuminated region induced by the first and the secondsubstantially narrow spectral band sources; and comparing intensities ofthe respective autofluorescence emission to assess in real-time, atleast one parameter selected from: tissue properties, the metabolicstate, injury, and tissue property changes resulting from therapeutic orprophylactic intervention of the tissue component region.

Another aspect of the present invention provides an apparatus forassessing tissue parameters that includes inducing autofluorescence in asame tissue component region with a source beam area of greater thanabout 1.5 mm² and comparing intensities of recorded autofluorescenceemission from the same tissue component region to assess in real-time,at least one parameter selected from: tissue properties, the metabolicstate, injury, and tissue property changes resulting from therapeutic orprophylactic intervention of the tissue component region.

Still another aspect of the present invention is an apparatus forassessing tissue parameters that includes inducing autofluorescence in asame tissue component region with a laser source beam area of greaterthan about 1.5 mm². Such a source can be directed by an optical fiberbundle having a sheath for registering tissue components with the distalend of the fiber bundle, wherein intensities of recordedautofluorescence received by the fiber bundle from the same tissuecomponent region are compared to assess in real-time, at least oneparameter selected from: tissue properties, the metabolic state, injury,and tissue property changes resulting from therapeutic or prophylacticintervention of the tissue component region.

Accordingly, the present provides optical arrangements and methods,capable of directing predetermined spectral radiation and capable ofproviding received spectral information for the assessment in real-timeof desired tissue parameters. Applications include, but are not limitedto, assessment of tissue viability in clinical environments, rapidassessment of tissue viability in civilian and battlefield traumascenarios, solid organ transplant, cerebrovascular disease,cardiovascular disease, diabetic tissue injury and ischemic boweldisease.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of the specification, illustrate specific embodiments of theinvention and, together with the general description of the inventiongiven above, and the detailed description of the specific embodiments,serve to explain the principles of the invention.

FIG. 1 shows a simplified diagram of diagnostic apparatus to determinedesired tissue parameters.

FIG. 2 shows an enlarged view of an example radiation conduit of thepresent invention.

FIGS. 3 a and 3 b, illustrates real-time injured (i.e., kidney tissue ina state of ischemia) to normal (control kidney tissue) autofluorescenceintensity measurements.

FIG. 4 shows intensity data measurements from about 240 nm to about 340nm.

FIGS. 5 a-d shows a series of fluorescence images collected by thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings, specific embodiments of the invention areshown. The detailed description of the specific embodiments, togetherwith the general description of the invention, serves to explain theprinciples of the invention.

General Description

The present invention provides apparatus and methods for real-timedetermination and quantification of the state of tissue components. Ingeneral, the apparatus as disclosed herein, can include an optical probehaving at least one illumination optical fiber, a plurality ofcollection optical fibers, at least one radiation source so as to causescattering and/or photoexcitation so as to induce autofluorescence in atargeted tissue component; a detector system that acquires suchautofluorescence and scattered spectra of polarized and unpolarizedlight; and a processor primarily arranged for comparing receivedautofluorescence signals so as to assess in real-time tissue properties,the metabolic state, injury, and tissue property changes resulting fromtherapeutic or prophylactic intervention of tissue components, such as,but not limited to brain, kidney, liver, heart, bowel, stomach, skin,pancreas, and muscle.

Specific Description

Turning now to the drawings, a diagram that illustrates an exemplaryembodiment of a system constructed in accordance with the presentinvention is shown in FIG. 1. The system, designated generally by thereference numeral 10, and capable of being designed as a portablecompact apparatus, includes the following basic components: a processor2, such as, but not limited to, a desktop computer, a laptop computer, aPersonal Digital Assistant (e.g. a handheld personal computer), or anyprocessing unit capable of handling the data of the present invention;one or more sources of electromagnetic radiation 4, 8 having a knownintensity and having an emission wavelength of at least 250 nm; opticalshutters 12, 16; optical element 20; and a radiation conduit 24 (e.g.,an optical fiber bundle) adapted to direct transmitted and collectedinduced autofluorescence and/or a scattered desired radiation fromtissue components 32 through one or more optical filters 44, such asedge filters, band-pass filters, polarization filters, and/or notchfilters, to allow desired bands and polarized components ofelectromagnetic radiation from tissue components 32 to be recorded byone or more detectors 48.

Electromagnetic radiation sources 4, 8 can be configured to emit aspectral band along paths H, I (shown with accompanying arrows), uponactivation of electronically or mechanically driven shutters 12, 16, asshown in FIG. 1. Although radiation sources 4, 8 can include abroad-band white light lamp capable of emitting filtered excitationradiation to interact with desired tissue components 32 (e.g., by longpass, band-pass, narrow-band pass filters, etc.), such sources are moreoften a monochromatic optical source, such as, a pulsed and/or a CW(continuous wave) laser, having desired power output levels of at least1 mW, and often having wavelengths of at least 250 nm, more often awavelength range between about 260 nm and about 360 nm, and even moreoften a 360 nm and/or a 335 nm wavelength.

Although several commercially available types of laser systems can beemployed in practice of the invention, an exemplary system oftenincludes an optical parametric oscillator, (i.e., a nonlinear materialcapable of producing a coherent beam of light that can be tuned over awide range of wavelengths) to obtain the required power levels andwavelengths as required. However, any lasing apparatus, such as diodelasers, dye lasers, tripled Nd:Yag systems, etc., having sufficientspectral bandwidth and power levels, and geometry to be integrated withthe present invention may be utilized without departing from the spiritand scope of the present invention.

Optical element 20, such as, for example, an e-beam depositedbeam-splitter, a liquid crystal splitter, an electro-optic device, anacousto-optic device, a mechanically driven reflective devices, and/or apredetermined dichroic mirror, is arranged to receive the radiation anddirect such radiation along a beam path denoted by the letter L, alsoshown with an accompanying arrow.

Directed radiation L is then received by a proximal end 22 of aradiation conduit 24, e.g., an optical fiber bundle that includes apredetermined illumination optical fiber 28 and one or more collectionoptical fibers 30 a-30 g housed in a cable. Laser radiation L passesthrough fiber 28 and is directed through to a distal end 26 so as tosubstantially and uniformly illuminate (shown as dashed arrows anddenoted by the letter L) a desired tissue component 32 region with anillumination area of at least greater than 1.5 mm². Optical fibers 30a-30 g are configured to collect scattered and/or inducedautofluorescence (shown as solid arrows and denoted by the letter F)resulting from radiation L and are directed back through fibers 30 a-30g, toward proximal end 22 and redirected along a routed path 36 througha filter 44, (shown as letter K) such as, for example, an analyzingpolarizer such that parallel, linear polarization, orthogonalcross-polarization, orthogonal elliptical polarization, same ellipticalpolarization, opposite circular polarization, or non-polarizationanalysis of the autofluorescence emission or scattered radiation F maybe employed.

In addition, a spectral pass filter, such as, but not limited to, a longpass filter to ensure a proper spectral band selection between about 290nm and about 1000 nm can also be positioned as filter 44 to collect adesired spectral radiation. Upon such filtering, the collected radiationcan be recorded by detector 48, such as, for example, a photodiode, aphotomultiplier, a spectrometer, a pixilated imaging sensor, a chargecoupled device (CCD), and/or any imaging device constructed to thedesign output parameters for system 10 and processed in real-time by,for example, a computer 2 in determining and quantifying a desiredtissue component and/or organ state.

FIG. 2 shows another example radiation conduit, i.e., an optical fiberassembly, generally designated as reference numeral 100, in more detail.As illustrated, laser illumination L (as shown by the enlargeddirectional arrow) can be directed through optical fiber 28, shownenclosed in a housing 29 with adjacent collection optical fibers 30 a-30g, so as to illuminate tissue 32. The collected scattered and/or inducedfluorescence radiation F then passes in a backscattered geometry,through optical fibers 30 a-30 g each having, for example, a 100 microncore diameter with predetermined separation gaps of at least about 0.20mm so as to direct received radiation to detector 48, as discussedabove, and as shown in FIG. 1.

A beneficial feature of the arrangement shown in FIG. 2 is a sheath 50,configured to position distal end 26 (shown as a dashed line) of opticalfibers 28 and 30 a-30 g with respect to tissue 32 by bringing in toregistry sheath 50 with tissue 32 (shown at dashed line 58). Moreover,sheath 50, can be configured with an ultra violet transmissive window52, disposed within interior wall 56 to enable wavelengths greater thanabout 190 nm to be transmitted by fiber 28 for illumination of tissue 32components. In addition, window 52 operates with sheath 50 to encloseand protect optical fibers 28 and 30 a-30 g from tissue contamination.Moreover, sheath 50 can be arranged with one or more additional opticalelements (not shown) disposed within interior wall 56, such as,diffractive optical components (lenses) and/or filters (polarizationfilters, long-pass filters, band-pass filters, etc.) to efficientlydeliver desired beam characteristics and radiation properties (e.g.,bandwidth and polarization) from sources 4, 8, to tissue components 32.

An additional beneficial example feature of sheath 50 is a reflectivesurface 60 to radiation induced from tissue 32, wherein a ray pathoutside of the acceptance angle of collection fibers 30 a-30 g, such asray F′, as shown in FIG. 2, can be internally reflected and collectedby, for example, fiber 30 b. Such a reflective feature can be produced,for example, by having sheath 50 arranged with a deposited reflectivecoating 56, such as, by vacuum deposition, CVD, e-beam deposition, orany method known to those skilled in the art capable of producing such acoating or by for example, manufacturing sheath 50 from a material, suchas a metal and having its interior wall polished so as to increasecollection efficiency for predetermined wavelengths induced byillumination wavelength L.

The combined investigative approach method embodiment of polarized lightscattering and/or biological fluorophore autofluorescence ratios usingthe apparatus, as discussed above, includes the following techniques.

Autofluorescence

Fluorophores in biological tissue, such as but not limited to,tryptophan, collagen, elastin, and NAD (nicotinamide adeninedinucleotide) emit fluorescence when illuminated with radiation. NAD, inparticular, is at much lower concentrations within tissue components,such as in humans, and consequently provides for low level emissionintensity when subjected to an excitation wavelength relative to otherfluorophores.

However, the intrinsic mitochondrial fluorophore, nicotinamide adeninedinucleotide (NADH), exhibits a peak in its emission spectrum at about470-nm when excited at a wavelength within its absorption spectrum,e.g., 335-nm, and is therefore likely the molecule responsible for thechanging spectroscopic properties associated with certain tissue statesin humans or animals, e.g., ischemia and hypoxia. During cellularrespiration, NAD is reduced to NADH to serve as an electron transporterin the oxygen dependent process that regenerates the cellular energyunit ATP. Without oxygenated blood flow, NADH accumulates in tissue andcauses an increase in autofluorescence when excited with the appropriatewavelength. Therefore, fluorescence from probed, for example, “injured”tissue components having such a fluorophore results in a rise inintensity of a fluorescence spectrum resulting from an increase in theinduced NADH. Unlike NADH, certain predetermined biologicalfluorophores, such as, tryptophan, is not directly involved in aerobicor anaerobic cellular respiration. Accordingly, the spectroscopicproperties of tryptophan is “inert”, i.e., induced autofluorescence isinsensitive to tissue parameters, such as, but not limited to, ischemia.

The present invention provides a beneficial use of such fluorescenceproperties by comparing NADH fluorescence emission induced by anabsorbing wavelength within the NADH spectrum to the relativefluorescence of a predetermined fluorophore, e.g., Tryptophan, producedby an absorbing wavelength within such a flurophore's absorptionspectrum.

Specifically, by comparing autofluorescence intensity at 335-nm, whichchanges with tissue property states, such as a tissue component in thestate of ischemia, with autofluorescence of, for example, tryptophan,induced by 260-nm excitation in the same area, parameters, such astissue properties, the metabolic state, injury, and tissue propertychanges resulting from therapeutic or prophylactic intervention ofprobed tissue components can be realized in real-time, e.g., less thanabout a minute.

Another beneficial arrangement is to produce standards frompredetermined tissue components based on, for example, but not limitedto, a persons age, sex, weight, and race to be stored by a processor orother means so as to normalize compared signals as discussed above andenhance the analysis capabilities of the present invention.

Elastic Light Scattering

Another example embodiment of the present invention is the incorporationof polarized elastic light scattering to additionally assess tissuecharacteristics to allow an end-user to acquire clinical diagnosticdeep-subsurface (e.g., at least 1 cm) images in collaboration with theautofluorescence method as discussed above. An illumination wavelengthfrom an electromagnetic radiation source can be utilized to provide apredetermined mean photon penetration depth larger. Linearcross-polarization and spectral analysis of the scattered photonssubstantially removes the photon information from the orthogonalillumination polarization resulting from the surface and allowssubstantially all of the scattered photons from the subsurface tissue tobe imaged.

In addition, a spectral polarization difference technique (SPDI) can beutilized in the present invention. Such a similar method is disclosed inpending U.S. application Ser. No. 10/190,231, titled “Near InfraredSpectroscopic Tissue Imaging In Medical Applications,” by Demos et al.,the disclosure which is herein incorporated by reference in itsentirety. With SPDI, different illumination wavelengths are utilized torecord, for example, images having a differential mean photonpenetration depth. Thus, smaller differential illumination wavelengthscan provide a narrower differential depth zone while a larger differencein two exemplary illuminating wavelengths gives rise to a wider depthzone. Cross-polarization and normalization analysis coupled withinter-image operations such as but not limited to subtraction betweenone or more illuminating wavelengths can provide information as to thetissue structure between the penetration depths of the one or morerespective probe illumination wavelengths. The present invention usessuch a technique in addition to comparing autofluorescence and lightscattering intensity measurements, such as single wavelengthcross-polarized light scattered measurements from the same predeterminedtissue component regions for analysis of tissue parameters as discussedherein.

An exemplary prototype apparatus and experiments were constructed andperformed at the UC Davis Medical Center in Sacramento, Calif. Thefollowing data are used to only illustrate some of the novelcapabilities of the present invention.

FIGS. 3 a and 3 b, illustrates capabilities of the present inventionwherein real-time injured (i.e., kidney tissue in a state of ischemia)to normal (control kidney tissue) autofluorescence intensity ratios aremeasured. Such measurements includes 335 nm excitation imaged through a450 nm narrow band filter, of in-vivo kidneys made ischemic 72(symbolized as circles) for 20 minutes, as shown in FIG. 3 a, and 90minutes, as shown in FIG. 3 b and subsequently reperfused (symbolized assquares).

Specifically, FIGS. 3 a and 3 b, illustrates real-time, in-vivoexperiments in anesthetized rats with exposed kidneys. Injured kidneysunderwent clamping to cause injury 72 of their vascular pedicles for 20minutes, as shown in FIG. 3 a or 90 minutes, as shown in FIG. 3 b,followed by unclamping and a recovery phase 74. Kidneys ischemic for 20minutes exhibited a recovery rebound of intensity toward a baseline 78,as shown in FIG. 3 a, whereas kidneys injured for 90, as shown in FIG. 3b, minutes did not exhibit any recovery of intensity.

FIG. 4 illustrates expansion of the autofluorescence techniques of thepresent invention using other excitation wavelengths in the UV spectralregion (i.e., from about 240 nm to about 340 nm). Specifically, FIG. 4shows Injured to normal intensity ratios 80 (symbolized as triangles),using various excitation wavelengths and imaged through a 395 long passfilter, for kidneys undergoing 60 minutes of ischemic damage. At 260 nmexcitation, the injured to normal intensity ratio is 1.

In effect, FIG. 4 illustrates a novel and beneficial embodiment of thepresent invention wherein an injured kidney appears inert when analyzedby 260 nm. Thus, instead of using an injured to uninjured intensityratio, as shown in FIGS. 3 a and 3 b, as a measurement of ischemia in aninjured kidney, 335 nm to 260-nm excitation ratio from an injured kidneyalone, as shown in FIG. 4, can be utilized as a method for real-timeassessment of the state of a tissue component and/or organ. By utilizingsuch a comparison method, coupled with techniques described herein, thepresent invention can rapidly assess tissue parameters as stated above.

FIGS. 5 a, 5 b, 5 c and 5 c further illustrates the method of thepresent invention with a set of injured and normal kidney intensityimages taken through a 395 long-pass filter under 335-nm and 260-nmexcitation. As predicted by the data in FIG. 4, obvious differences inintensity due to 20 minutes, as shown in 5 a and 5 c, and 90 minutes asshown in 5 b and 5 d, of ischemic injury under 335-nm excitation, asshown in 5 a and 5 b, disappear under 260-nm excitation, as shown in 5 cand 5 d.

Another beneficial method of the present invention involves monitoringfluorescing contrast agents infused in various organs and/or tissue byarterial circulation in situ using the methods and apparatus of thepresent invention. Alternatively, such contrast agents can be infusedin, for example, an organ, through a perfusion pump. The amount of suchcontrast agents remaining in a tissue component region or organ after agiven period of time is proportional to the amount of capillarypermeability and thus to the degree of the state of a tissue, e.g.,ischemia. Using techniques and apparatus described herein coupled withutilized contrast agents provides a quantifiable measurement of desiredtissue parameters.

Still another beneficial arrangement of the present invention is toilluminate with a set of wavelengths, preferably two, and image apredetermined component region. The objective is to employ hyperspectral(i.e., using various spectroscopic techniques and multiple wavelengthand/or spectral bands) imaging to investigate the ability of polarizedlight in combination with predetermined tissue componentautofluorescence so as to also assess in real-time, tissue properties,the metabolic state, injury, and tissue property changes resulting fromtherapeutic or prophylactic intervention of said tissue componentregion.

Applicants are providing this description, which includes drawings andexamples of specific embodiments, to give a broad representation of theinvention. Various changes and modifications within the spirit and scopeof the invention will become apparent to those skilled in the art fromthis description and by practice of the invention. The scope of theinvention is not intended to be limited to the particular formsdisclosed and the invention covers all modifications, equivalents, andalternatives falling within the spirit and scope of the invention asdefined by the claims.

1. A method for assessing tissue parameters, comprising: illuminating adesired tissue component region with a first substantially narrowspectral band source; illuminating said desired tissue component regionwith a second substantially narrow spectral band source, wherein saidilluminated region by said first and said second substantially narrowspectral band sources comprises an area of greater than about 1.5 mm²;detecting a respective autofluorescence emission from said desiredilluminated region induced by said first and said second substantiallynarrow spectral band sources; and comparing intensities of saidrespective autofluorescence emission to assess in real-time at least oneparameter selected from: tissue properties, the metabolic state, injury,and tissue property changes resulting from therapeutic or prophylacticintervention of said tissue component region.
 2. The method of claim 1,wherein said autofluorescence emission from said tissue component regioncomprises a spectral region between about 400 nm and about 1000 nm. 3.The method of claim 1, wherein said autofluorescence emission from saidtissue component region comprises a spectral region between about 290 nmand about 800 nm.
 4. The method of claim 1, wherein saidautofluorescence comprises an emission from a biological fluorophore. 5.The method of claim 1, wherein said first and said second substantiallynarrow spectral band sources comprise a monochromatic light source. 6.The method of claim 5, wherein said monochromatic light source can emita wavelength of at least 250 nm.
 7. The method of claim 5, wherein saidfirst substantially narrow spectral band source comprises a wavelengthwithin the absorption spectrum of NADH.
 8. The method of claim 5,wherein said second substantially narrow spectral band source comprisesa wavelength within the absorption spectrum of tryptophan.
 9. The methodof claim 1, wherein a source for said first and/or said secondsubstantially narrow spectral bands comprises a broad band lamp designedto transmit predetermined filtered wavelengths.
 10. The method of claim1, wherein said illuminating steps comprises directing said first andsaid substantially narrow spectral bands via an optical fiber bundle soas to substantially illuminate an area having a long dimension of up toabout 10 cm.
 11. The method of claim 10, wherein said optical fiberbundle is adapted for receiving a scattered radiation and saidautofluorescence.
 12. The method of claim 1, wherein said detecting stepcomprises hyperspectral microscopy to image tissue microstructure andassess microscopic alterations associated with tissue injury.
 13. Themethod of claim 1, wherein said tissue component region is in a state ofischemia.
 14. The method of claim 1, wherein said tissue componentregion is in a state of hypoxia.
 15. The method of claim 1, wherein saidfirst and second substantially narrow spectral band sources arepolarized and said autofluorescence emission from said tissue componentregion is analyzed.
 16. The method of claim 1, wherein said first andsecond substantially narrow spectral band sources are linearly polarizedand a parallel-polarized component of said autofluorescence emission isanalyzed.
 17. The method of claim 16, wherein a perpendicularlypolarized component of said autofluorescence emission is analyzed. 18.The method of claim 1, wherein said autofluorescence emission having anopposite circular polarization orientation with respect to saidsubstantially narrow spectral band sources is analyzed.
 19. The methodof claim 1, wherein said first and said second substantially narrowspectral band sources are elliptically polarized and a same ellipticalpolarization orientation of said autofluorescence emission is analyzed.20. The method of claim 19, wherein an orthogonal ellipticalpolarization of said autofluorescence emission is analyzed.
 21. Themethod of claim 1, wherein said first and second substantially narrowspectral band sources are polarized and a scattered electromagneticradiation is analyzed.
 22. The method of claim 21, wherein an orthogonalpolarization component of said scattered electromagnetic radiation isanalyzed.
 23. The method of claim 1, wherein said detecting stepcomprises a charge coupled device.
 24. The method of claim 1, whereinsaid detecting step comprises a device selected from a photomultiplierand a photodiode.
 25. The method of claim 1, wherein said injured anduninjured tissue components comprise at least one tissue componentselected from brain, kidney, liver, heart, bowel, skin, stomach,pancreas, and muscle.
 26. The method of claim 1, wherein a fluorescingcontrast agent can be infused into said tissue components to determine aquantifiable measurement of tissue injury.
 27. An apparatus forassessing tissue parameters, comprising: a source of electromagneticradiation configured to produce at least two predetermined substantiallynarrow spectral bands; a radiation source conduit adapted for directingsaid substantially narrow spectral bands so as to illuminate a sametissue component region with a source beam area of greater than about1.5 mm² and induce autofluorescence, wherein said conduit isadditionally adapted for receiving and directing said autofluorescence;and a device adapted to record said received autofluorescence from saidtissue component region; and means for comparing intensities of saidrecorded autofluorescence emission from said same tissue componentregion to assess in real-time, at least one parameter selected from:tissue properties, the metabolic state, injury, and tissue propertychanges resulting from therapeutic or prophylactic intervention of saidtissue component region.
 28. The apparatus of claim 27, wherein saidsource comprises one or more lasers having wavelengths at least greaterthan 250 nm.
 29. The apparatus of claim 27, wherein said source furthercomprises one or more commercially available compact lasers.
 30. Theapparatus of claim 29, wherein said compact lasers comprise laserdiodes.
 31. The apparatus of claim 27, wherein said source comprises abroad band optical lamp designed to transmit predetermined filteredwavelengths.
 32. The apparatus of claim 27, wherein saidautofluorescence emission from said tissue component region comprises aspectral band between about 400 nm and about 1000 nm.
 33. The apparatusof claim 27, wherein said autofluorescence emission from said tissuecomponent region comprises a spectral band between about 290 nm andabout 800 nm.
 34. The apparatus of claim 27, wherein saidautofluorescence comprises an emission from a biological fluorophore.35. The apparatus of claim 27, wherein said substantially narrowspectral bands comprise a wavelength within the absorption spectrum ofNADH.
 36. The apparatus of claim 27, wherein said substantially narrowspectral bands comprise a wavelength within the absorption spectrum oftryptophan.
 37. The apparatus of claim 27, wherein hyperspectralmicroscopy can be utilized to image tissue microstructure and assessmicroscopic alterations associated with said injured tissue components.38. The apparatus of claim 27, wherein said tissue component region isin a state of ischemia.
 39. The apparatus of claim 27, wherein saidtissue component region is in a state of hypoxia.
 40. The apparatus ofclaim 27, wherein said first and second substantially narrow spectralbands are polarized and said autofluorescence emission from said sametissue component region is analyzed.
 41. The apparatus of claim 27,wherein said first and second substantially narrow spectral bands arelinearly polarized and a parallel-polarized component of saidautofluorescence emission from said same tissue component region isanalyzed.
 42. The apparatus of claim 41, wherein a perpendicularlypolarized component of said autofluorescence emission from said sametissue component region is analyzed.
 43. The apparatus of claim 27,wherein said autofluorescence emission having an opposite circularpolarization orientation with respect to said substantially narrowspectral bands is analyzed.
 44. The apparatus of claim 27, wherein saidfirst and said second substantially narrow spectral bands areelliptically polarized and a same elliptical polarization orientation ofsaid autofluorescence emission is analyzed.
 45. The apparatus of claim44, wherein an orthogonal elliptical polarization of saidautofluorescence emission is analyzed.
 46. The apparatus of claim 27,wherein said first and second substantially narrow spectral bands arepolarized and a scattered electromagnetic radiation is capable of beinganalyzed.
 47. The apparatus of claim 46, wherein an orthogonalpolarization component of said scattered electromagnetic radiation isanalyzed.
 48. The apparatus of claim 27, wherein said device comprises acharge coupled device.
 49. The apparatus of claim 27, wherein saiddevice comprises at least one detector selected from a photomultiplierand a photodiode.
 50. The apparatus of claim 27, wherein said sametissue component region comprises at least one tissue component selectedfrom brain, kidney, liver, heart, bowel, stomach, skin, pancreas, andmuscle.
 51. The apparatus of claim 27, wherein said source conduitcomprises an optical fiber bundle.
 52. The apparatus of claim 51,wherein said fiber bundle comprises at least one illumination fiberhaving a diameter between about 500 microns and about 1 mm.
 53. Theapparatus of claim 51, wherein said fiber bundle comprises one or moreoptical fibers for receiving a scattered radiation and saidautofluorescence.
 54. The apparatus of claim 52, wherein saidillumination fiber is arranged to illuminate a tissue area having a longdimension of up to about 10 cm.
 55. An apparatus for assessing tissueparameters, comprising: one or more laser sources having predeterminedwavelengths; an optical fiber bundle adapted for directing said lasersources from a proximal end to produce autofluorescence at a distal endin a same tissue component region and additionally adapted for receivingsaid autofluorescence at said distal end; a protective sheath configuredon said optical fiber bundle and adapted for positioning said distal endof said optical fiber bundle with respect to said tissue components andadditionally adapted for redirecting said produced autofluorescenceoutside of the acceptance angle of said distal end of said optical fiberbundle so as to additionally be received for recordation; a deviceadapted to record said received autofluorescence from said tissuecomponents; and means for comparing intensities of said recordedautofluorescence from said same tissue component region to assess inreal-time, at least one parameter selected from: tissue properties, themetabolic state, injury, and tissue property changes resulting fromtherapeutic or prophylactic intervention of said tissue componentregion.
 56. The apparatus of claim 55, wherein said laser sourcescomprise wavelengths at least greater than 250 nm.
 57. The apparatus ofclaim 55, wherein said sources further comprise one or more commerciallyavailable compact lasers.
 58. The apparatus of claim 55, wherein saidsources further comprise a diode monochromatic light source.
 59. Theapparatus of claim 55, wherein said sources comprise a broad bandoptical lamp designed to transmit predetermined filtered wavelengths.60. The apparatus of claim 55, wherein said autofluorescence from saidinjured tissue comprises a spectral region between about 400 nm andabout 1000 nm.
 61. The apparatus of claim 55, wherein saidautofluorescence from said uninjured tissue comprises a spectral regionbetween about 290 nm and about 800 nm.
 62. The apparatus of claim 55,wherein said autofluorescence comprises an from a biologicalfluorophore.
 63. The apparatus of claim 55, wherein at lest one of saidlaser sources comprises a wavelength within the absorption spectrum ofNADH.
 64. The apparatus of claim 55, wherein at least one of said lasersources comprises a wavelength within the absorption spectrum oftryptophan.
 65. The apparatus of claim 55, wherein hyperspectralmicroscopy can be utilized to image tissue microstructure and assessmicroscopic alterations associated with said injured tissue components.66. The apparatus of claim 55, wherein said tissue component region isin a state of ischemia.
 67. The apparatus of claim 55, wherein saidtissue component region is in a state of hypoxia.
 68. The apparatus ofclaim 55, wherein said laser sources comprise a polarized output andsaid autofluorescence from said same tissue component region isanalyzed.
 69. The apparatus of claim 55, wherein said laser sourcescomprise a linearly polarized output and a parallel-polarized componentof said autofluorescence from said same tissue component region isanalyzed.
 70. The apparatus of claim 69, wherein a perpendicularlypolarized component of said autofluorescence from said same tissuecomponent region is analyzed.
 71. The apparatus of claim 55, whereinsaid autofluorescence having an opposite circular polarizationorientation with respect to said laser sources output is analyzed. 72.The apparatus of claim 55, wherein said laser sources are ellipticallypolarized and a same elliptical polarization orientation of saidautofluorescence is analyzed.
 73. The apparatus of claim 72, wherein anorthogonal elliptical polarization of said autofluorescence is analyzed.74. The apparatus of claim 55, wherein an output of said laser sourcesis polarized and a scattered electromagnetic radiation is capable ofbeing analyzed.
 75. The apparatus of claim 74, wherein an orthogonalpolarization component of said scattered electromagnetic radiation isanalyzed.
 76. The apparatus of claim 55, wherein said device comprises acharge coupled device.
 77. The apparatus of claim 55, wherein saiddevice comprises at least one detector selected from a photomultiplierand a photodiode.
 78. The apparatus of claim 55, wherein said injuredand uninjured tissue components comprise at least one tissue componentselected from brain, kidney, liver, heart, bowel, stomach, skin,pancreas, and muscle.
 79. The apparatus of claim 55, wherein saidoptical fiber bundle comprises at least one illumination fiber having adiameter between about 500 microns and about 1 mm.
 80. The apparatus ofclaim 79, wherein said at least one illumination fiber is arranged toilluminate a tissue area having a long dimension of up to about 10 cm.81. The apparatus of claim 55, wherein said optical fiber bundlecomprises one or more optical fibers for receiving a scattered radiationand said autofluorescence.
 82. The apparatus of claim 55, wherein saidprotective sheath further comprises a material adapted for transmittingultra violet.
 83. The apparatus of claim 55, wherein said protectivesheath further comprises a reflective material to enable the redirectionof said produced autofluorescence outside of the acceptance angle ofsaid optical fiber bundle.
 84. The apparatus of claim 83, wherein saidreflective material comprises a polished metal.
 85. The apparatus ofclaim 83, wherein said reflective material comprises a reflectivecoating.